1. Field
The field is electrophoretic separations, and in particular to their visualization and analysis.
2. Prior Art—Protein Separations—FIGS. 1-2
The individual components of protein mixtures, such as those with different molecular weights, often must be separated in order to determine the relative quantities of each within the mixture. Separations of the proteins within cells are useful in comparing diseased and healthy tissue samples, for example. In the past, such components were separated according to their molecular weight using gel-electrophoresis in well-known fashion. A number of gel materials were used to elute or draw out the components according to their molecular weights. One such gel was polyacrylamide. A clear gel 100, as shown in FIG. 1, typically had an area about 100 cm2 and thickness 1.0 mm. Smaller and larger, thicker and thinner gels have been used. Other gels and methods have been used to separate amino acids and other compounds.
After separation, gel 100 was generally transparent and devoid of color. Protein components 105 typically reside within lanes, one of which is indicated by a dashed outline 110. Several means have been used to visualize the as yet invisible separated protein components 105, indicated by dashed lines. In one method of visualization, components 105 are made visible by immersing the gel in a stain such as Comassie blue, silver, and others.
After staining, FIG. 2, previously invisible components 105 (FIG. 1) were visible and were seen as darker regions 120 within gel 100. Gel 100 absorbed very little of the stain. The position of each of regions 120 was analyzed to reveal the molecular weight of the corresponding component 105. Their extent and optical density revealed the volume of protein component 105 within each band.
Instead of staining after separation, stains, fluorescent materials, or radioactive tags can be applied to components 105 prior to separation.
While staining is well-known, it is cumbersome and time-consuming. Radioactive tagging presents a safety and disposal hazard. Incorporating a stain or fluorescent molecule prior to separation can change the molecular weight of the material being analyzed, thereby causing errors in analysis. In addition, gels are very fragile and they must be handled carefully during staining and analysis, or they will separate or tear.
Prior-Art—Dielectric Constant Measurement—FIGS. 3-5
A brief discussion about electrical measurements used in the present invention follows. The dielectric constant of a volume of insulating or semi-insulating material 300 (FIG. 3) can be measured using an electrical circuit. Electrodes 305 and 310 are placed in contact with material 300. A voltage source 315 is connected to electrode 310. The inverting input of an operational amplifier 320 is connected to electrode 305. The non-inverting input of amplifier 320 is connected to ground return, as is the second terminal of source 315. The output terminal 319 of amplifier 320 is connected to the vertical input, indicated by “V” in FIG. 3, of an oscilloscope 330. The horizontal time base, “H”, of oscilloscope 330 is triggered by the output of source 315. Resistor 340 and capacitor 350 determine the gain of amplifier 320 in well-known fashion. With resistor 340 and capacitor 350 in place, the inverting input of amplifier 320 is maintained at ground potential in the circuit shown. Linear operation of amplifier 320 is assumed. Instead of oscilloscope 330, other circuitry can be used including a peak detector, microprocessor, and the like, in well known fashion.
Assume that the volume of material 300 is 0.5 mm thick between electrodes 305 and 310, and that its area is 2 mm2. Neglecting edge effects, the capacitance of the volume of material 300 is given by the formula below.
  C  =            ɛ      ⁢                          ⁢      A        d  ∈ is the dielectric constant of volume 300, A is the area of electrodes 305 and 310, and d is the thickness of volume 300. ∈ is generally represented as the product of ∈o, the permittivity of vacuum, and ∈r, the relative dielectric constant of the material under study. The value of ∈0 is 8.85×10−12 Farad/m. If ∈r of the material in volume 300 is 20, then Cv=7×10−1 picofarads (pf).
If source 315 provides a square wave, as shown in FIG. 4, the output of amplifier 320 will be a transient peak, followed by a decay, as shown in FIG. 5. The rate of decay is determined by resistor 340 and capacitor 350. The associated time constant is equal to the product of the values of resistor 340 and 350. If the value of resistor 340 is one megohm (1 MΩ), and the value of capacitor 350 is 1 pf, then the decay time constant is one microsecond (μS). If a period of ten time constants is allowed to elapse between pulses, then the maximum frequency of the applied square wave is 50 kHz.
If the volume in material 300 is insulating, the steady-state gain of amplifier 320 will be unity. The transient gain is given by the ratio of the capacitance of the material in volume 300 and the capacitance of capacitor 350. If the value of capacitor 350 is 5 pf, then the gain of amplifier 320 is 0.7 pf/5 pf=0.14. Therefore if the voltage output of source 315 is 10 volts (V) peak-to-peak (p-p), the output of amplifier 320 will be 1.4 V p-p.
Electrical Conductivity Measurement—FIGS. 3, 4, and 6
The above circuitry can be used to measure the electrical conductivity of the volume in material 300. Instead of measuring the peak-to-peak value as described above (FIG. 5), the direct-current (DC) value or asymptote is measured (FIG. 6). At the asymptote the current density in material 300 is given byj=σE,where j=the current density in amperes per square cm, σ=the conductivity in Siemens/mm (S/mm), and E is the amplitude of the electric field applied between electrodes 305 and 310 in V/mm.
As above, assume that the volume of material 300 is 0.5 mm thick between electrodes 305 and 310, that its area is 2 mm2. Further, assume that the applied voltage is 10 V, and that the asymptotic value of the current (FIG. 6) is 10−9 A. The conductivity of material 300 is thus 0.5×10−10 S/mm.
In summary, FIG. 4 represents the output of source 315. FIG. 5 represents the output of amplifier 320 when the electrical properties of the contents of volume 300 are mainly capacitive. FIG. 6 represents the output of amplifier 320 when the contents of volume 300 have a mixture of capacitive and resistive properties.
Instead of using square-wave voltage source 315, a sinusoidal source (not shown) can be used in well-known fashion. The mathematics used in analysis of signals excited by a sinusoidal source is described in detail in my U.S. Pat. No. 6,265,883 (2001) which is incorporated herein by reference.